1. Field of the Invention
The present invention concerns a laser manipulation apparatus for trapping a micro-sample selected from a group of micro-sample s dispersed and suspended in a liquid medium under the view field of a microscope and separating the trapped sample from other micro-sample s.
2. Statement Related Art
For instance, Escherichia coli are often used as micro-sample s for genetic manipulation. In this case, the genetic manipulation is applied by dispersing the samples in a predetermined liquid medium, then Escherichia coli dispersed in the liquid medium are sucked together with the liquid medium by a micropipette or the like, transferred into and cultured in a culture vessel and a great amount of DNA are reproduced.
However, not all of the Escherichia coli dispersed in the liquid medium are genetically manipulated because of the individual difference, and a plurality of Escherichia coli are sucked simultaneously into a micropipette, so that cultured Escherichia coli comprise mixture of those with and without genetic manipulation and the yield for those applied with the genetic manipulation is low.
If culture from a single Escherichia coli is possible, pure culture is enabled and only Escherichia coli subjected to genetic manipulation can be obtained at 100% yield if the starting single Escherichia coli was genetically manipulated.
When it is intended to take out only one of micro-sample s such as biological particles or micro-particles under the view field of an optical microscope, there is no substantial problem if the size of the samples is somewhat large. However, if it is too small as Escherichia coli, since the view field is restricted in view of the optical property of lenses, it is extremely difficult to take out the aimed sample while microscopically confirming that other micro-sample s are not mixed.
For instance, in a case of taking-out a biological particle of 100 .mu.m length, it can be taken-out by using a micropipette of 200 .mu.m diameter while observing that other biological particles are not mixed under a microscope having a lens with a resolution power of 10 .mu.m, a focal depth of 300 .mu.m, NA=0.034 and a view field diameter of 10 mm. On the other hand, in a case of taking-out a biological particle of only about 1 .mu.m length such as Escherichia coli sucked together with 1 mm.sup.3 of a liquid medium into a micropipette, it can not be confirmed that other biological particles are not mixed even if a lens used has a resolution power of 0.26 .mu.m and NA=1.3 (oil-immersed objective lens having 100 magnification factor), which are the limit of an optical lens, because the focal depth is only 0.2 .mu.m and the view field is only 200 .mu.m.
Further, even if the micro-sample has such a size as can be confirming by a microscope, if a great number of biological particles or like other particles are present in 1 mm.sup.3 of a liquid medium to be sucked into a micropipette, the great number of such individuals may possibly be sucked simultaneously or deposited at the top end of the micropipette and mixed upon dropping of the liquid medium. After all, although it is possible to take-out a great number of biological particles simultaneously by sucking a great amount of a liquid medium, it is difficult to take out only one of biological particles or like other particles, and this enforces troublesome repeating diluting operation.
Further, even if the density of micro-sample s present in the liquid medium is low, those moving at a relatively high speed such as Escherichia coli (several .mu.m to several tens .mu.m/s) are difficult to be trapped since the operation of a micropipette following after the movement results in stirring of the liquid medium to flow the biological particles by the stream.
It has been proposed, in U.S. Pat. No. 5,100,627, a chamber comprising sample supply channels for flowing a liquid medium in which micro-sample s are dispersed and sample separation channels for flowing a liquid medium in which no micro-sample s are dispersed. They are formed in parallel with each other, and each of the channels is in communication by an interconnection channel. The interconnection channel is conducted intermittently by a bubble valve thereby taking out the micro-sample flowing through the sample supply channel only by one to the sample separation channel.
In this bubble valve, a bubble channel for flowing a bubble-containing liquid medium is formed in parallel with the sample supply channel and the sample separation channel and crossing the interconnection channel, so that the interconnection channel is conducted by filling a liquid medium to the intersection between the bubble channel and the interconnection channel and shut by situating the bubble.
However, in this structure, it is extremely difficult to control the pressure balance between the liquid medium supplied by the bubble channel and the liquid medium filled in the sample supply channel and the sample separation channel.
For instance, when the bubble or the liquid medium is supplied by the bubble channel, since the liquid medium in other channels flows under the effect thereof, it is extremely difficult and not practical to carry out on-off control only for the interconnection channel as desired with no effect on other channels.
Meanwhile, in a case of optically trapping a biological particle, trapping has been conducted by using an IR laser as a light source (refer to Japanese Patent Publication Hei 5-6136, on the basis of the priority of U.S. Pat. No. 4,893,886).
This is based on the result of experiment by Ashkin, et al that light-induced biological damages were observed when biological particles were trapped by using a visible light laser (Ar laser: wavelength at 514 nm) as a light source, whereas no light-induced biological damages were observed when biological samples are trapped by using an IR laser (YAG laser: wavelength at 1064 nm) as a light source.
However, the optical trap by the IR laser involves the following problems compared with the optical trap by the visible light.
At first, since the visible light can be seen, a danger, if caused by entrance of light to an eye, can be avoided by closing an eye lid or interrupting the light, whereas the IR light can not be seen and is liable to damage eyes with no awareness for the entrance of light.
Secondly, since the visible light can be recognized visually, adjustment for the optical system is easy when the apparatus is set, whereas the IR light can not be recognized visually and, therefore, adjustment for the optical system is difficult.
Thirdly, a trapping force is generally weak as a wavelength is longer. For instance, for a polyethylene latex particle of 3 .mu.m size, the trapping force of a YAG laser (IR light: wavelength at 1064 nm) is smaller by 25% than the trapping force of a red light (visible light: wavelength at 600 nm). In the case of 1 .mu.m particle size, the trapping force of the YAG laser is smaller by 45% as compared with the trapping force of the red light.
Fourthly, optical trapping is conducted at a focal position of an objective lens (converging optical system), a beam spot diameter at the focal position is in proportion with a wavelength and the beam spot diameter increases as the wavelength is longer, so that it is more difficult to trap a small sample by the IR light as compared with the visible light.
Fifthly, general-purpose parts for usual microscopes such as lenses, mirrors and other optical elements are used for the laser manipulation apparatus in order to reduce the cost. Since they are designed for the light in the visible light region, if a light in the IR region is used, the optical transmittance is decreased to reduce the efficiency of utilizing the laser light, as well as aberration is increased to lower the light convergence.
For example, in a case of an objective lens of 100 magnification factor, optical transmittance is more than 90% for the red light (visible light: wavelength at 600 nm), whereas the optical transmittance for the YAG laser (IR light: wavelength at 1064 nm) is lowered to less than 30%. Accordingly, in a case of using the IR light, a light source having a power about three times as large as that of the visible light has to be used if it is intended to irradiate a light at a same level of intensity.
Further, in a case of using the IR light, since the aberration of the lens is large, the beam spot diameter at the focal position increases to reduce the trapping force for a small sample.
The foregoings are concerned with the matters of lens design. Accordingly, it is of course technically possible to design such that IR transmittance is improved or aberration is reduced. Then, if optical lenses adapted exclusively for the IR light are used, the problems for optical transmittance or aberration can be overcome. However, in a case of design and manufacture of such products for exclusive use, it results in another problem of increasing the manufacturing cost to increase the cost for the entire apparatus.
The IR light has been used for optical trapping of biological particles irrespective of various drawbacks compared with the visible light, because it is highly demanded for separating a single sample alive, for example, in a case of culturing Escherichia coli applied with genetic manipulation.
In a case of using the technique of optical trapping for taking out a single Escherichia coli, there is a premise that the Escherichia coli is taken out alive as it is. An identical conclusion is also derived from the study of Sakano, et al in addition to Ashkin described above (articles of Static Electricity Society/October, 1991).
According to Sakano, et al, it was reported that when an Ar laser (wavelength at 514 nm) was irradiated to Escherichia coli at 0.64 mW//.mu.m.sup.2, movement stopped in about 7 sec, whereas the movement did not stop even if irradiation was conducted by applying a YAG laser (wavelength at 1064 nm) for more than two hours at a double intensity of 1.28 mW/.mu.m.sup.2.
Then, the light-induced damages were compared between the case of 514 nm and 1064 nm wavelength, and it was concluded that the wavelength in the IR region is more safe as compared with the wavelength in the visible light regarding the light-induced damages on biological particles. This denies the possibility of using light at a wavelength in the visible light region for trapping the biological samples
However, if Escherichia coli can be trapped alive by using a light at a wavelength in the visible light region, various advantages are obtainable compared with the use of a light in the IR light region.
In view of the above, the present inventors, et al have studied on a relationship between the wavelength of a laser light irradiated to biological particles and light-induced damages on the biological particles.
At first, according to the study of F. Bryant (Archives of Biochemistry and Biophysics, vol. 135/1969: Absolute Optical Cross Sections of Cells and Chloroplasts), it is reported that the spectral absorption of Escherichia coli or yeast bacteria varies in the visible light region and that this is not attributable to the light absorption of bacteria but to the scattering of light by bacteria.
When the present inventors, et al made a similar experiment for a light in a wider wavelength range of 150 to 1100 nm by a spectrophotometer using an integrating sphere, it was confirmed that the spectral absorbance changes in accordance with the wavelength as shown by a solid line in FIG. 11 and it was confirmed that Escherichia coli or yeast have large light absorption to the light at a wavelength of shorter than 300 nm, the light absorption is extremely small for the light in a longer wavelength region up to 1100 nm and there is a scarce difference in the absorption depending on the wavelength.
The wavelength of the light of an Ar laser (visible light: wavelength at 514 nm) at which biological damages were observed by the report of Ashkin, et al or Sakano et al is within a region of 300 to 1100 nm that does not result in light absorption upon irradiation on Escherichia coli, etc. and could cause no biological damages. However, since biological damages are actually observed, it is assumed that the biological damages are not caused by linear usual light absorption.
The power density of 0.64 mW/.mu.m.sup.2 (=640,000 kW/m.sup.2) of the light used in the experiment by Sakano, et al is 640,000 times as high as the power density of 1 kW/m.sup.2 of sun light irradiated on the ground in fine weather. In the field of light at such a high density, various non-linear interactions are taken place between the light and the substance irradiated therewith and two photon absorption is most likely to occur among them. The two photon absorption is the absorption caused in proportion with the square of the light intensity and does not occur at usual light intensity.
D. Dinkel, et al reported that two photon absorption was observed when a light at 580 nm was irradiated on yeast (Analytica Chemica Acta, 236/1992, Remote Two-Photon Excited Fluorescence Sensing in a Simulated Fermentation Broth). In this experiment, tryptophan as a sort of essential amino acids constituting proteins of yeast absorbed two photons at 580 nm, and took an identical excited state as if they were irradiated with UV-rays at 290 nm, i.e., one-half wavelength of 580 nm.
It can be considered that biological damages were observed for the Ar laser (visible light: wavelength at 514 nm) in the report of Ashkin, et al and Sakano, et al, not because the wavelength was in the visible light region but because the two photon absorption phenomenon resulted in the same state as that irradiated by a light at 257 nm which is one-half wavelength of 514 nm.
In view of the above, the present inventors, et al have come to a conclusion that, in a case of optically trapping biological particles such as Escherichia coli or yeast bacteria by a laser light, biological damages are cause in a case of using a wavelength from 150 nm to 300 nm that is absorbed by proteins or nucleic acids constituting them (FIG. 11; shown by solid line) and in a case of using a laser light having a wavelength from 300 nm and 600 nm that is absorbed by two photon absorption (FIG. 11; shown by dotted line).
Biological particles include those having dyes and not having dyes. Those not having dyes such as Escherichia coli, yeast bacteria and paramecium do not cause light absorption by the dyes, but those having dyes such as Euglenida, corspuscles and photosynthesis bacteria absorb light of particular colors depending on dyes thereof and, in this case, even the IR light which is considered not to cause biological damages by the existent experiment may cause biological damages.